Project Summary Periodontal disease affects nearly 50% of adults in the United States, negatively impacting oral function and the quality of life of those adults affected. Porphyromonas gingivalis (Pg) is an oral anaerobe strongly implicated in the etiology of adult periodontal disease due, in part, to its ability to colonize and persist within the subgingival biofilm. Therefore, understanding how biofilm development is mediated is of fundamental importance to understanding the pathogenic potential of Pg. Removal of arginine by bacterial arginine deiminases inhibits Pg biofilm development by down-regulating fimbriae. The genome of Pg does not encode an arginine deiminase, but it does encode a peptidylarginine deiminase (Porphyromonas peptidylarginine deiminase or PPAD) that preferentially converts peptidylarginine to peptidylcitrulline while also converting free L-arginine to L-citrulline at a slower rate. Although it has been shown that arginine deiminases inhibit Pg biofilm formation by removing arginine, the impact of PPAD on Pg biofilm formation remains a significant gap in knowledge. Therefore, this study aims to investigate PPAD and its effects on Pg biofilm development. Preliminary data indicates that a deletion of the gene encoding PPAD enhances biofilm formation by Pg; however, the enhanced biofilm is not due to an up-regulation of fimbriae, indicating that PPAD activity modulates surface attached growth by a novel mechanism. Given the preliminary data, the central hypothesis is that PPAD is a regulated enzyme that modulates biofilm development via citrullination of arginine residues within select peptides and/or proteins. This hypothesis will be tested through two specific aims: 1) Characterize PPAD expression and activity, and 2) Determine how PPAD activity modulates Pg biofilm formation. In Aim 1, PPAD expression throughout growth under typical culture conditions will be determined by measuring mRNA levels, protein levels, and enzymatic activity. Using this information as a baseline, the effects of temperature, hemin availability, oxidative stress, and substrate availability via arginine gingipain (Rgp) activity on PPAD expression and activity will be determined. In Aim 2, biofilms of WT Pg will be compared to those of the PPAD deletion mutant in terms of morphology, viability, and matrix composition. Additionally, western blots and mass spectrometry will be used to identify citrullinated proteins in order to better understand how PPAD activity modulates biofilm development. A better understanding of the regulatory mechanisms that control Pg biofilm formation will contribute to the development of intervention strategies for the treatment and prevention of periodontal disease. In particular, PPAD may be a novel therapeutic target for the treatment and prevention of periodontal disease and associated inflammatory diseases. Furthermore, this research represents the basis of a doctoral dissertation project. Hence, this fellowship application will assist in training a student with an interest in oral health research to become an independent investigator.